Human induced pluripotent stem cells to unravel the pathophysiology of peripartum cardiomyopathy

Peripartum Cardiomyopathy (PPCM) is a pregnancy-associated myocardial disease
characterized by signs and symptoms of heart failure (HF). The etiology of PPCM
is largely unknown. Using tissue of patients with PPCM may be helpful in
unraveling the pathophysiology of the disease. However, in human this is
challenged by the fact that myocardial biopsies are taken only in a minority of
patients. The procedure is associated with the risk of myocardial perforation
and gives only a limited amount of tissue. Furthermore, if biopsies are taken,
this is mostly done in patients with already far advanced disease, and
therefore the early changes in cardiomyocyte structure and signaling pathways
remain unknown. Therefore a novel and innovative approach is needed.
With the use of human induced pluripotent stem cells (hiPSC) we will generate
patient-specific cardiomyocytes that will be subjected to pregnancy related
stress. This allows us to explore new pathways in PPCM. Finally, the influence
of the target genes and microRNA's identified in the hiPSC experiments will be
studied in relation to new onset HF in the general population.

Employing PPCM patient-derived iPSCs, we aim to test our hypothesis that
cardiomyocytes from PPCM patients exhibit (epi)genetically determined
differences in protein expression profiles and functional behavior in
comparison to those from healthy controls. We will therefore follow specific
aims: Aim 1: To isolate skin fibroblasts from PPCM patients and healthy control
individuals by punch biopsy under local anesthesia and store them after in
vitro multiplication.
Aim 2: To reprogram the patient-derived skin fibroblasts into induced
pluripotent stem cells (iPSCs) and, after full iPSC
characterization/verification, differentiate them in vitro into cardiomyocytes.
Aim 3: To compare the characteristics of PPCM patient-derived cardiomyocytes
with control ones using RNA sequencing, proteomic analyses and epigenomic
profiling.
Aim 4: To expose PPCM patient and control-derived cardiomyocytes to different
types of stress (stretch, oxidative, and hormonal) in vitro and compare their
responses by analyzing their behavior and expression profiles.
Aim 5: To correlate findings in iPS cells to bloodlevels of certain proteins
and microRNAs.

Non-therapeutic study, exploring 2 groups of 6 Dutch PPCM-patients and 2 groups
of healthy controls, one visit research.
For the whole study, fibroblasts from 2 groups of 6 PPCM patients and 2 groups
of 6 healthy controls will be used. One group of patients that suffered a
severe case of PPCM and one group of patients that have recovered from (mild)
PPCM will be selected. Each control group will consist of sisters of the PPCM
patients. When sisters are unavailable, nieces will be selected. In total, 24
subjects will be selected.
After patient selection, a skin punch biopsy (6mm) will be performed according
to standard procedures under sterile conditions and after local anesthesia at
the cardiology outpatient clinic, UMCG. The skin biopsies will be collected in
sterile physiological salt solution and transported to the lab of the dept. of
Experimental Cardiology, UMCG. After tissue dissociation, fibroblasts will be
cultured and multiplied under fibroblast specific/selective culture conditions.
Part of these fibroblasts will be stored frozen; another part will be used for
reprogramming into iPSCs and subsequently differentiated into cardiomyocytes
according to procedures developed in the dept. of Experimental Cardiology,
UMCG. Subsequently, the iPSC-derived cardiomyocytes will be subjected to
extensive analyses, comprising proteomics, gene expression and epigenomic
profiling. These characteristics will be compared with cardiomyocytes generated
from iPSC lines derived from healthy controls. Control iPSC lines will be
obtained from and equal number of healthy sisters. Healthy controls are all
women aged of 18 years and older with no signs of HF development. All included
subjects have to have been pregnant at least once.
In order to obtain baseline values, bloodsamples will be taken from all
individuals involved and an echocardiogram will be made. The bloodsamples will
be stored for future reference to which we can compare findings obtained from
the iPS cells; certain protein and microRNA levels will be determined from
those bloodsamples.

From the PPCM patients and healthy control subjects, a small (6mm) skin biopsy
will be taken from the inner side of the upper arm using a standard punch
technique under local anesthesia. The only minimal risk involved in
particpation may be the occurrence of infection (despite intense desinfection
of the site of biopsy) (<1% of cases) ; effective treatment with antibiotic
ointment.
The extent of risks involved in taken a blood sample is small. Minor
complications may occur as an hematoma or an infection. These complications are
very rare and blood samples will be taken by an expert.